C Frequency dependence of the action potential parameters of the atrial myocytes. Asterisks indicate significant differences between stimulus frequencies. Next, we assessed the effects of noradrenalin, acetylcholine, or a combination thereof on the action potentials of our SA nodal myocytes. As illustrated in Figure 2 A, application of noradrenalin resulted in an increase in the spontaneous beating frequency, associated with an increase in the diastolic depolarization rate without any striking differences in other action potential parameters.
Application of acetylcholine, on the other hand, resulted in a clear hyperpolarization of the maximum diastolic potential, a decrease in the diastolic depolarization rate and a dramatic increase in cycle length Figure 2 B.
If acetylcholine was applied in the presence of noradrenalin, there was no substantial hyperpolarization of the maximum diastolic potential and the increase in cycle length was less prominent Figure 2 C. Figure 2. Effects of noradrenalin, acetylcholine, or a combination of both on the action potentials of isolated rabbit SA nodal myocytes.
Figure 3 summarizes the effects on action potential parameters for a total of 13 cells. These data confirm the aforementioned effects on cycle length Figure 3 A , diastolic depolarization rate Figure 3 B , and maximum diastolic potential Figure 3 C.
In addition, Figure 3 shows that both noradrenalin and acetylcholine caused an increase in maximum upstroke velocity, which was larger in case of acetylcholine Figure 3 E. Figure 3 may suggest that acetylcholine increases action potential amplitude Figure 3 D and action potential duration Figures 3 F—H , but it should be noted that these data are not statistically significant.
Figure 3. Asterisks indicate significant differences between effects of noradrenalin and acetylcholine. Hashes indicate significant differences from control. A Change in cycle length. C Change in maximum diastolic potential MDP. D Change in action potential amplitude APA. We also assessed the effects of noradrenalin, acetylcholine, or a combination thereof on the action potentials of our atrial myocytes. The results are shown in Figures 4 — 7. Figure 4. Effects of noradrenalin, acetylcholine, or a combination of both on the action potentials of isolated rabbit left atrial myocytes.
As illustrated in Figure 4 A, application of noradrenalin resulted in a slight hyperpolarization of the maximum diastolic potential. The same was found for acetylcholine Figure 4 B , but not so much for the combination of noradrenalin and acetylcholine Figure 4 C.
In all cases, i. Effects on action potential duration differed between noradrenalin and acetylcholine, with an increase in case of noradrenalin and a decrease for acetylcholine Figures 4 A,B and a less pronounced effect for the combination of noradrenalin and acetylcholine Figure 4 C.
Figure 5 summarizes the effects on action potential parameters for a total of 23 cells. As already observed in the typical examples of Figure 4 , there is a slight hyperpolarization of the maximum diastolic potential Figure 5 A and an increase in action potential amplitude Figure 5 B. Also, under all conditions there is an increase in maximum upstroke velocity Figure 5 C. The effects on APD 50 and APD 90 are different between the three conditions, with a decrease in case of acetylcholine, an increase in case of noradrenalin and a smaller, but still significant increase in case of acetylcholine combined with noradrenalin Figures 5 E,F.
Figure 5. Asterisks indicate significant differences between effects of noradrenalin, acetylcholine, or a combination of both. A Change in maximum diastolic potential MDP. B Change in action potential amplitude APA. In addition to the experiments of Figures 4 and 5 , in which the cells were stimulated at 2 Hz, we carried out experiments in which we varied the stimulation frequency over the range of 1—4 Hz. The data, obtained from a total of 12 cells, did not reveal a clear-cut frequency dependence in the effects of acetylcholine or noradrenalin on action potential duration Figure 6.
Figure 6. Cells were stimulated at 1—4 Hz. Asterisk indicates significant difference between stimulus frequencies. In a total of five cells, we varied these concentrations over the range of 1— nM. However, it should be noted that the data with acetylcholine are from one cell, as are the data in the combined presence of noradrenalin and acetylcholine.
Figure 7. Concentration was varied between 1 and nM. Asterisks indicate significant differences between concentrations. Note logarithmic abscissa. In the present study, we have assessed the effects of noradrenalin, acetylcholine, or a combination thereof on the action potential configuration of rabbit SA nodal and left atrial myocytes.
Action potentials were recorded under close-to-physiological conditions. To the best of our knowledge, the present study is the first in which the effects of noradrenalin, acetylcholine, or a combination thereof on the action potential configuration of isolated sinoatrial and atrial myocytes has been tested in detail under such close-to-physiological conditions. As detailed in Figure 1 , our SA nodal and atrial myocytes showed clearly distinct action potential characteristics.
We obtained our myocytes from rabbit, which is a widely used animal model in the field of cardiac cellular electrophysiology. In many respects, human action potential morphology is better resembled by rabbit than by mouse Brunner et al. Yet, one should be aware that there may be species differences in the underlying mechanisms, as there are, e. The beating rate of our SA nodal cells increases upon application of noradrenalin and decreases upon application of acetylcholine Figures 2 A,B and 3 A , which is associated with an increase and a decrease in the rate of diastolic depolarization, respectively Figure 3 B.
However, one should be cautious when drawing conclusions regarding underlying mechanisms from changes in action potentials. This may be illustrated by the increase in maximum upstroke velocity that is observed in response to noradrenalin as well as acetylcholine Figure 3 E. In case of noradrenalin, this increase may well result from an upregulation of the L-type calcium current I Ca,L Hagiwara et al.
Additionally, the activation of a small number of fast sodium channels may have contributed to the increase in maximum upstroke velocity as a result of the acetylcholine-induced hyperpolarization of the maximum diastolic potential which results in incomplete voltage-dependent inactivation of these channels Veldkamp et al. A full exploration of the autonomic modulation of the complex dynamic interactions of voltage- and calcium-dependent processes within the SA nodal pacemaker cell may require in silico experiments Maltsev and Lakatta, Such experiments have already proven useful in a computer simulation study of the autonomic modulation of the electrical activity of bullfrog atrial myocytes Shumaker et al.
A reduction in beating frequency as well as a hyperpolarization of the maximum diastolic potential was also reported for SA nodal myocytes isolated from wild-type mice in response to carbachol Cifelli et al.
In the top panel of their Online Figure 5 , Yang et al. However, in contrast to our SA nodal myocytes, such hyperpolarization was also observed in response to noradrenalin. Furthermore, we found an increase in action potential amplitude and, as in our SA nodal myocytes, an increase in maximum upstroke velocity both in response to noradrenalin and in response to acetylcholine Figures 5 B,C.
This may be explained by an increase in the fast sodium current, which is responsible for the fast upstroke in atrial cells, due to the more negative maximum diastolic potential and thus larger availability of fast sodium channels less voltage-dependent inactivation. On the other hand, increased intracellular calcium levels in response to noradrenalin may have inhibitory effects on these sodium channels Casini et al. Action potential duration is increased in response to noradrenalin and decreased in response to acetylcholine, with more or less additive effects if both noradrenalin and acetylcholine are present Figures 5 D—F.
The shortening of the action potential in response to acetylcholine is in line with data from literature Koumi et al. Of note, acetylcholine does not inhibit the L-type calcium current of atrial myocytes under basal conditions, in contrast to that of SA nodal myocytes Petit-Jacques et al.
However, Yeh et al. Furthermore, a dose dependence in the effects of isoproterenol and phenylephrine on the action potential and the underlying transmembrane currents may play a role Shumaker et al.
The data on action potential duration of Figure 5 were obtained at a stimulus frequency of 2 Hz. Figure 6 demonstrates that similar effects were obtained at other frequencies in the range of 1—4 Hz, without a clear-cut frequency dependence, although the increase in APD 50 at 1 Hz in the presence of noradrenalin is significantly different from that at 4 Hz.
In the present study, we have used the amphotericin-perforated patch-clamp technique to test the effects of noradrenalin, acetylcholine, or a combination thereof on the action potential configuration of isolated sinoatrial and atrial myocytes under close-to-physiological conditions. Whereas this approach allows a detailed study of the effects of a well-controlled dose of these neurotransmitters on the intrinsic action potential configuration of these isolated myocytes, it does not take into account the complexity and heterogeneity of the intact myocardium.
Therefore, caution should be applied when translating our in vitro results to the in vivo situation. Furthermore, although human action potential morphology is better resembled by rabbit than by mouse, there may be species differences in the effects of noradrenalin and acetylcholine. Our data show that acetylcholine and noradrenalin can modulate the beating rate of SA nodal myocytes and the action potential duration of atrial myocytes over a wide range.
Name Emory University. Related projects. Am J Physiol C J Gen Physiol J Biol Chem Mol Pharmacol Hartzell, H C; Hirayama, Y; Petit-Jacques, J Effects of protein phosphatase and kinase inhibitors on the cardiac L-type Ca current suggest two sites are phosphorylated by protein kinase A and another protein kinase. Cardiovasc Drugs Ther 7 Suppl Frace, A M; Hartzell, H C Opposite effects of phosphatase inhibitors on L-type calcium and delayed rectifier currents in frog cardiac myocytes.
Dempsey, P. Entman, M. Cyclic Nucl. Fabiato, A. Fleckenstein A. In: Calcium and the heart Harris, P. London-New York: Academic Press George, W. Giles, W. Goldberg, N. Hashimoto, K. Tohoku J. Higgins, C. Hoffman, B. Hollenberg, M. Ikemoto, Y. Japan Acad. Inui, J. Naunyn-Schmiedeberg's Arch. Katz, A. Cyclic Nucl Res. Kukovetz, W. LaRaia, P. Lee, T.
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